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81.
Construction and characterization of Escherichia coli strains deficient in multiple secreted proteases: protease III degrades high-molecular-weight substrates in vivo. 总被引:6,自引:5,他引:1 下载免费PDF全文
Protease III, the product of the ptr gene, is a 110-kDa periplasmic protease with specificity towards insulin and other low-molecular-weight substrates (less than 7,000 molecular weight) in vitro (Y.-S.E. Cheng and D. Zipser, J. Biol. Chem. 254:4698-4706, 1979). Escherichia coli strains deficient in protease III were constructed by insertional inactivation of the ptr gene. This mutation did not appear to affect the function of the adjoining recB and recC genes. Expression of protein A-beta-lactamase, a protease-sensitive secreted polypeptide, was increased approximately twofold in ptr cells. A comparable increase in the half-life of protein A-beta-lactamase was observed by pulse-chase experiments, suggesting that protease III is involved in the catabolism of high-molecular-weight substrates in vivo, ptr mutants exhibited no detectable phenotypic alterations except for a slight reduction in growth rate. When the ptr mutation was transferred to a strain deficient in the secreted protease DegP, a further decrease in growth rate, as well as an additive increase in the expression of the fusion protein, was observed. A ptr degP ompT mutant strain resulted in a further increase in expression in minimal medium but not in rich medium. 相似文献
82.
Abnormal fractionation of beta-lactamase in Escherichia coli: evidence for an interaction with the inner membrane in the absence of a leader peptide. 下载免费PDF全文
beta-Lactamase with the -20 to -1 region of the leader peptide deleted (almost complete deletion of the leader peptide) [delta(-20,-1) beta-lactamase] was released from Escherichia coli cells by osmotic shock. Fractionation of the cells by conversion to spheroplasts and protease accessibility experiments further indicated that a portion of the protein may be bound to the cytoplasmic membrane and be partially exposed in the periplasmic space. Expression of delta(-20,-1) beta-lactamase conferred a 25-fold increase in the 50% lethal dose for ampicillin relative to that for controls, thus confirming that a small amount (about 2%) of the active protein is completely exported from the cytoplasm. These results suggest that even in the absence of a leader peptide, mature beta-lactamase is able to interact with the cytoplasmic membrane and be translocated into the periplasmic space, albeit with a low efficiency. 相似文献
83.
Aquatic Ecology - Potamon potamios populations have decreased significantly due to the degradation of its habitat caused by human activities, mainly the use of insecticides. Today, P. potamios is... 相似文献
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85.
McCaffrey K Boo I Tewierek K Edmunds ML Poumbourios P Drummer HE 《Journal of virology》2012,86(7):3961-3974
Hepatitis C virus glycoprotein E2 contains 18 conserved cysteines predicted to form nine disulfide pairs. In this study, a comprehensive cysteine-alanine mutagenesis scan of all 18 cysteine residues was performed in E1E2-pseudotyped retroviruses (HCVpp) and recombinant E2 receptor-binding domain (E2 residues 384 to 661 [E2(661)]). All 18 cysteine residues were absolutely required for HCVpp entry competence. The phenotypes of individual cysteines and pairwise mutation of disulfides were largely the same for retrovirion-incorporated E2 and E2(661), suggesting their disulfide arrangements are similar. However, the contributions of each cysteine residue and the nine disulfides to E2 structure and function varied. Individual Cys-to-Ala mutations revealed discordant effects, where removal of one Cys within a pair had minimal effect on H53 recognition and CD81 binding (C486 and C569) while mutation of its partner abolished these functions (C494 and C564). Removal of disulfides at C581-C585 and C452-C459 significantly reduced the amount of E1 coprecipitated with E2, while all other disulfides were absolutely required for E1E2 heterodimerization. Remarkably, E2(661) tolerates the presence of four free cysteines, as simultaneous mutation of C452A, C486A, C569A, C581A, C585A, C597A, and C652A (M+C597A) retained wild-type CD81 binding. Thus, only one disulfide from each of the three predicted domains, C429-C552 (DI), C503-C508 (DII), and C607-C644 (DIII), is essential for the assembly of the E2(661) CD81-binding site. Furthermore, the yield of total monomeric E2 increased to 70% in M+C597A. These studies reveal the contribution of each cysteine residue and the nine disulfide pairs to E2 structure and function. 相似文献
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D. Brent Halling Dimitra K. Georgiou D. J. Black Guojun Yang Jennifer L. Fallon Florante A. Quiocho Steen E. Pedersen Susan L. Hamilton 《The Journal of biological chemistry》2009,284(30):20041-20051
Calmodulin binds to IQ motifs in the α1 subunit of CaV1.1 and CaV1.2, but the affinities of calmodulin for the motif and for Ca2+ are higher when bound to CaV1.2 IQ. The CaV1.1 IQ and CaV1.2 IQ sequences differ by four amino acids. We determined the structure of calmodulin bound to CaV1.1 IQ and compared it with that of calmodulin bound to CaV1.2 IQ. Four methionines in Ca2+-calmodulin form a hydrophobic binding pocket for the peptide, but only one of the four nonconserved amino acids (His-1532 of CaV1.1 and Tyr-1675 of CaV1.2) contacts this calmodulin pocket. However, Tyr-1675 in CaV1.2 contributes only modestly to the higher affinity of this peptide for calmodulin; the other three amino acids in CaV1.2 contribute significantly to the difference in the Ca2+ affinity of the bound calmodulin despite having no direct contact with calmodulin. Those residues appear to allow an interaction with calmodulin with one lobe Ca2+-bound and one lobe Ca2+-free. Our data also provide evidence for lobe-lobe interactions in calmodulin bound to CaV1.2.The complexity of eukaryotic Ca2+ signaling arises from the ability of cells to respond differently to Ca2+ signals that vary in amplitude, duration, and location. A variety of mechanisms decode these signals to drive the appropriate physiological responses. The Ca2+ sensor for many of these physiological responses is the Ca2+-binding protein calmodulin (CaM).2 The primary sequence of CaM is tightly conserved in all eukaryotes, yet it binds and regulates a broad set of target proteins in response to Ca2+ binding. CaM has two domains that bind Ca2+ as follows: an amino-terminal domain (N-lobe) and a carboxyl-terminal domain (C-lobe) joined via a flexible α-helix. Each lobe of CaM binds two Ca2+ ions, and binding within each lobe is highly cooperative. The two lobes of CaM, however, have distinct Ca2+ binding properties; the C-lobe has higher Ca2+ affinity because of a slower rate of dissociation, whereas the N-lobe has weaker Ca2+ affinity and faster kinetics (1). CaM can also bind to some target proteins in both the presence and absence of Ca2+, and the preassociation of CaM in low Ca2+ modulates the apparent Ca2+ affinity of both the amino-terminal and carboxyl-terminal lobes. Differences in the Ca2+ binding properties of the lobes and in the interaction sites of the amino- and carboxyl-terminal lobes enable CaM to decode local versus global Ca2+ signals (2).Even though CaM is highly conserved, CaM target (or recognition) sites are quite heterogeneous. The ability of CaM to bind to very different targets is at least partially due to its flexibility, which allows it to assume different conformations when bound to different targets. CaM also binds to various targets in distinct Ca2+ saturation states as follows: Ca2+-free (3), Ca2+ bound to only one of the two lobes, or fully Ca2+-bound (4–7). In addition, CaM may bind with both lobes bound to a target (5, 6) or with only a single lobe engaged (8). If a target site can bind multiple conformers of CaM, CaM may undergo several transitions that depend on Ca2+ concentration, thereby tuning the functional response. Identification of stable intermediate states of CaM bound to individual targets will help to elucidate the steps involved in this fine-tuned control.Both CaV1.1 and CaV1.2 belong to the L-type family of voltage-dependent Ca2+ channels, which bind apoCaM and Ca2+-CaM at carboxyl-terminal recognition sites in their α1 subunits (9–14). Ca2+ binding to CaM, bound to CaV1.2 produces Ca2+-dependent facilitation (CDF) (14). Whether CaV1.1 undergoes CDF is not known. However, both CaV1.2 and CaV1.1 undergo Ca2+- and CaM-dependent inactivation (CDI) (14, 15). CaV1.1 CDI is slower and more sensitive to buffering by 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid than CaV1.2 CDI (15). Ca2+ buffers are thought to influence CDI and/or CDF in voltage-dependent Ca2+ channels by competing with CaM for Ca2+ (16).The conformation of the carboxyl terminus of the α1 subunit is critical for channel function and has been proposed to regulate the gating machinery of the channel (17, 18). Several interactions of this region include intramolecular contacts with the pore inactivation machinery and intermolecular contacts with CaM kinase II and ryanodine receptors (17, 19–22). Ca2+ regulation of CaV1.2 may involve several motifs within this highly conserved region, including an EF hand motif and three contiguous CaM-binding sequences (10, 12). ApoCaM and Ca2+-CaM-binding sites appear to overlap at the site designated as the “IQ motif” (9, 12, 13), which are critical for channel function at the molecular and cellular level (14, 23).Differences in the rate at which 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid affects CDI of CaV1.1 and CaV1.2 could reflect differences in their interactions with CaM. In this study we describe the differences in CaM interactions with the IQ motifs of the CaV1.1 and the CaV1.2 channels in terms of crystal structure, CaM affinity, and Ca2+ binding to CaM. We find the structures of Ca2+-CaM-IQ complexes are similar except for a single amino acid change in the peptide that contributes to its affinity for CaM. We also find that the other three amino acids that differ in CaV1.2 and CaV1.1 contribute to the ability of CaV1.2 to bind a partially Ca2+-saturated form of CaM. 相似文献
89.
Evangelos J. Giamarellos-Bourboulis Maria Raftogiannis Anastasia Antonopoulou Fotini Baziaka Pantelis Koutoukas Athina Savva Theodora Kanni Marianna Georgitsi Aikaterini Pistiki Thomas Tsaganos Nikolaos Pelekanos Sofia Athanassia Labrini Galani Efthymia Giannitsioti Dimitra Kavatha Flora Kontopidou Maria Mouktaroudi Garyfallia Poulakou Vissaria Sakka Periklis Panagopoulos Antonios Papadopoulos Kyriaki Kanellakopoulou Helen Giamarellou 《PloS one》2009,4(12)
Background
The pandemic by the novel H1N1 virus has created the need to study any probable effects of that infection in the immune system of the host.Methodology/Principal Findings
Blood was sampled within the first two days of the presentation of signs of infection from 10 healthy volunteers; from 18 cases of flu-like syndrome; and from 31 cases of infection by H1N1 confirmed by reverse RT-PCR. Absolute counts of subtypes of monocytes and of lymphocytes were determined after staining with monoclonal antibodies and analysis by flow cytometry. Peripheral blood mononuclear cells (PBMCs) were isolated from patients and stimulated with various bacterial stimuli. Concentrations of tumour necrosis factor-alpha, interleukin (IL)-1beta, IL-6, IL-18, interferon (FN)-alpha and of IFN-gamma were estimated in supernatants by an enzyme immunoassay. Infection by H1N1 was accompanied by an increase of monocytes. PBMCs of patients evoked strong cytokine production after stimulation with most of bacterial stimuli. Defective cytokine responses were shown in response to stimulation with phytohemagglutin and with heat-killed Streptococcus pneumoniae. Adaptive immune responses of H1N1-infected patients were characterized by decreases of CD4-lymphocytes and of B-lymphocytes and by increase of T-regulatory lymphocytes (Tregs).Conclusions/Significance
Infection by the H1N1 virus is accompanied by a characteristic impairment of the innate immune responses characterized by defective cytokine responses to S.pneumoniae. Alterations of the adaptive immune responses are predominated by increase of Tregs. These findings signify a predisposition for pneumococcal infections after infection by H1N1 influenza. 相似文献90.
Emmanuel Dialynas Pantelis Topalis John Vontas Christos Louis 《PLoS neglected tropical diseases》2009,3(6)